Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices
Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices
Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices
An important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of
significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure
susceptibility to drugs of choice for particular infections. The most widely used testing methods include broth microdilution
or rapid automated instrument methods that use commercially marketed materials and devices. Manual methods that provide
flexibility and possible cost savings include the disk diffusion and gradient diffusion methods. Each method has strengths
and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results
(eg, minimum inhibitory concentration), and all provide qualitative assessments using the categories susceptible, intermediate,
or resistant. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms.
However, newer or emerging mechanisms of resistance require constant vigilance regarding the ability of each test method
to accurately detect resistance.
made broth dilution testing practical and popular. Standard ent diffusion method uses the principle of establishment of an
trays contain 96 wells, each containing a volume of 0.1 mL that antimicrobial concentration gradient in an agar medium as a
allows approximately 12 antibiotics to be tested in a range of means of determining susceptibility. The Etest (bioMérieux AB
8 two-fold dilutions in a single tray [2, 4]. Microdilution panels BIODISK) (Figure 2) is a commercial version available in the
are typically prepared using dispensing instruments that aliquot United States. It employs thin plastic test strips that are im-
precise volumes of preweighed and diluted antibiotics in broth pregnated on the underside with a dried antibiotic concentra-
into the individual wells of trays from large volume vessels. tion gradient and are marked on the upper surface with a
Hundreds of identical trays can be prepared from a single mas- concentration scale. As many as 5 or 6 strips may be placed in
ter set of dilutions in a relatively brief period. Few clinical a radial fashion on the surface of an appropriate 150-mm agar
microbiology laboratories prepare their own panels; instead fro- plate that has been inoculated with a standardized organism
zen or dried microdilution panels are purchased from one of suspension like that used for a disk diffusion test. After over-
several commercial suppliers. The cost of the preprepared pan- night incubation, the tests are read by viewing the strips from
els range from approximately $10 to $22 each. Inoculation of the top of the plate. The MIC is determined by the intersection
panels with the standard 5 ⫻ 10 5 CFU/mL is accomplished us- of the lower part of the ellipse shaped growth inhibition area
ing a disposable device that transfers 0.01 to 0.05 mL of stan- with the test strip.
dardized bacterial suspension into each well of the microdi- The gradient diffusion method has intrinsic flexibility by
lution tray or by use of a mechanized dispenser. Following being able to test the drugs the laboratory chooses. Etest strips
incubation, MICs are determined using a manual or automated cost approximately $2–$3 each and can represent an expensive
viewing device for inspection of each of the panel wells for approach if more than a few drugs are tested. This method is
growth [2]. best suited to situations in which an MIC for only 1 or 2 drugs
The advantages of the microdilution procedure include the is needed or when a fastidious organism requiring enriched
generation of MICs, the reproducibility and convenience of medium or special incubation atmosphere is to be tested (eg,
having preprepared panels, and the economy of reagents and penicillin and ceftriaxone with pneumococci) [5–7]. Generally,
space that occurs due to the miniaturization of the test. There Etest results have correlated well with MICs generated by broth
is also assistance in generating computerized reports if an au- or agar dilution methods [5–9]. However, there are some sys-
tomated panel reader is used. The main disadvantage of the tematic biases toward higher or lower MICs determined by the
microdilution method is some inflexibility of drug selections Etest when testing certain organism-antimicrobial agent com-
available in standard commercial panels. binations [6, 10]. This can represent a potential shortcoming
Antimicrobial gradient method. The antimicrobial gradi- when standard MIC interpretive criteria derived from broth
dilution testing [10] are applied to Etest MICs that may not The advantages of the disk method are the test simplicity
be identical. that does not require any special equipment, the provision of
Disk diffusion test. The disk diffusion susceptibility categorical results easily interpreted by all clinicians, and flex-
method [2, 11, 12] is simple and practical and has been well- ibility in selection of disks for testing. It is the least costly of
standardized. The test is performed by applying a bacterial all susceptibility methods (approximately $2.50–$5 per test for
inoculum of approximately 1–2 ⫻ 10 8 CFU/mL to the surface materials). The disadvantages of the disk test are the lack of
of a large (150 mm diameter) Mueller-Hinton agar plate. Up mechanization or automation of the test. Although not all fas-
to 12 commercially-prepared, fixed concentration, paper an- tidious or slow growing bacteria can be accurately tested by
tibiotic disks are placed on the inoculated agar surface (Figure this method, the disk test has been standardized for testing
3). Plates are incubated for 16–24 h at 35C prior to deter- streptococci, Haemophilus influenzae, and N. meningitidis
mination of results. The zones of growth inhibition around through use of specialized media, incubation conditions, and
each of the antibiotic disks are measured to the nearest mil- specific zone size interpretive criteria [12].
limeter. The diameter of the zone is related to the susceptibility Automated instrument systems. Use of instrumentation
of the isolate and to the diffusion rate of the drug through the can standardize the reading of end points and often produce
agar medium. The zone diameters of each drug are interpreted susceptibility test results in a shorter period than manual read-
using the criteria published by the Clinical and Laboratory ings because sensitive optical detection systems allow detection
Standards Institute (CLSI, formerly the National Committee of subtle changes in bacterial growth. There are 4 automated
for Clinical Laboratory Standards or NCCLS) [13] or those instruments presently cleared by the FDA for use in the United
included in the US Food and Drug Administration (FDA)– States. Three of these can generate rapid (3.5–16 h) suscepti-
approved product inserts for the disks. The results of the disk bility test results, while the fourth is an overnight system [16].
diffusion test are “qualitative,” in that a category of suscepti- The MicroScan WalkAway (Siemens Healthcare Diagnostics) is
bility (ie, susceptible, intermediate, or resistant) is derived from a large self-contained incubator/reader device that can incubate
the test rather than an MIC. However, some commercially- and analyze 40–96 microdilution trays. The WalkAway utilizes
available zone reader systems claim to calculate an approximate standard size microdilution trays that are hydrated and inoc-
MIC with some organisms and antibiotics by comparing zone ulated manually and then placed in one of the incubator slots
sizes with standard curves of that species and drug stored in in the instrument. The instrument incubates the trays for the
an algorithm [14, 15]. appropriate period, examining them periodically with either a
photometer or fluorometer to determine growth development. lution plates that can be inoculated with a Sensititre
Gram-negative susceptibility test panels containing fluorogenic Autoinculator. Growth is determined by fluorescence mea-
substrates can be read in 3.5–7 h. Separate gram-positive and surement after 18–24 h of incubation. Test panels are available
gram-negative panels read using turbidimetric end points are for gram-positive and gram-negative bacteria, S. pneumoniae,
ready in 4.5–18 hours. Haemophilus species, and nonfermentative gram-negative
The BD Phoenix Automated Microbiology System (BD Di- bacilli.
agnostics) has a large incubator reader with a capacity to process The Phoenix, Sensititre ARIS 2X, Vitek 1 and 2, and
99 test panels that contain 84 wells devoted to antibiotic dou- WalkAway instruments have enhanced computer software used
bling dilutions and are inoculated manually. The Phoenix mon- to interpret susceptibility results including “expert systems” for
itors each panel every 20 min using both turbidometric and analyzing test results for atypical patterns and unusual resis-
colorimetric (oxidation-reduction indicator) growth detection. tance phenotypes [16]. Two studies [17, 18] have shown that
Test panels for gram-negative, gram-positive, S. pneumoniae,
providing rapid susceptibility test results can lead to more
b-hemolytic, and viridans group streptococci are available. MIC
timely changes to appropriate antimicrobial therapy, substantial
results are generated in 6–16 h.
direct cost savings attributable to ordering of fewer additional
The Vitek 2 System (bioMérieux) is highly automated and
laboratory tests, performance of fewer invasive procedures, and
uses very compact plastic reagent cards (credit card size) that
a shortened length of stay. These benefits are best realized when
contain microliter quantities of antibiotics and test media in a
coupled with extended laboratory staffing schedules, and real-
64-well format. The Vitek 2 employs repetitive turbidimetric
time, electronic transmission of verified results. One of the early
monitoring of bacterial growth during an abbreviated incu-
bation period. The instrument can be configured to accom- shortcomings of rapid susceptibility testing methods was a less-
modate 30–240 simultaneous tests. The susceptibility cards al- ened ability to detect some types of antimicrobial resistance
low testing of common, rapidly growing gram-positive, and including inducible b-lactamases and vancomycin resistance.
gram-negative aerobic bacteria, and S. pneumoniae in a period However, the recently FDA-cleared instruments have made sig-
of 4–10 h. An older, less automated, Vitek 1 System is still used nificant improvements in large part through modifications of
in some laboratories. The system is more limited with a 45- the instruments’ computer software to either provide extended
well card and does not include S. pneumoniae. incubation for problematic organism-drug combinations, or by
The Sensititre ARIS 2X (Trek Diagnostic Systems) is an au- editing of susceptibility results using expert software to prevent
tomated, overnight, incubation and reading system with a 64- unlikely results from being reported. In some cases these mod-
panel capacity. The test panels are standard 96-well microdi- ifications result in prolonged incubation (ie, 110 h) of test